| Glioma is the most aggressive primary tumor in the central nervous system(CNS),accounting for more than 60%of adult brain tumors,and there is a high degree of heterogeneity among different patients.At present,the mainstream therapy for glioma still relies on surgical resection,and the use of radiotherapy,chemotherapy with temozolomide as adjuncts.However,due to the strong invasiveness of gliomas and the high rate of disease recurrence,the overall prognosis of patients after treatment is still poor,with the median survival time for only 14-16 months.CD59 is a type of complement regulatory protein anchored on the cell membrane by glycosylphosphatidylinositol(GPI),it inhibits the binding of C5b-8 and C9 to limit the formation of membrane attack complexes(MAC).Tumor cells can protect themselves from complement-mediated attacks through the high expression of CD59,and the function of immune cells in the tumor microenvironment will be affected by CD59.Therefore,tumor immunotherapy as an anti-cancer treatment strategy has attracted more and more attention.In this study,two glioma cell lines U87MG and U251MG were used as the research objects,the cells were transfected with short hairpin ribonucleic acid(shRNA)using lentivirus as a vector to knock down CD59 or transfect the coding sequence(CDS)to overexpress CD59,and use CD59 neutralizing antibody and membrane anchor signal blocker to block CD59 protein expression to observe the proliferation of glioma cells;Further through we chose Affymetrix gene chip to detect cells,data prediction analysis through ingenuity pathway analysis(IPA)and RT-PCR to verify gene expression,we preliminarily speculate that CyclinD family members and CD59 interact with each other,both of which affect the proliferation of glioma cells.The main research contents and results are as follows:1.The proliferation of glioma cellsWe used CD59-silencing lentivirus to infect glioma cells,and used RTCA X CELLigence to detect cell proliferation,supplemented by LIVE\DEAD staining and AnnexinV staining.The results showed that the proliferation of glioma cells was inhibited,and no apoptosis or cell necrosis occurred,suggesting that the down-regulation of CD59 may affect the proliferation of glioma cells.And after silencing CD59,the cell cycle was blocked in the G1/S phase.2.Block the expression of CD59We use CD59 neutralizing antibodies and membrane anchoring signal blockers to block CD59 protein function,use CD59 overexpressing lentivirus to infect glioma cells to upregulate CD59,and detect CD59 expression by RT-PCR.The results showed that cell proliferation was not inhibited after blocking the protein function of CD59,but after changing the expression of CD59 mRNA,cell proliferation changed with mRNA expression,Up-regulation of mRNA accelerated cell proliferation,and down-regulation of mRNA inhibited cell proliferation.Based on the above results,we believe that the proliferation of glioma cells is regulated by the expression level of CD59 mRNA.3.The mechanism of CD59 regulating the proliferation of glioma cellsWe used Affymetrix gene chip to detect cells,and analyzed data through IPA to screen out cyclin genes that are significantly related to CD59 down-regulation.We found that down-regulation of CD59 significantly affects the G1/S phase signal pathway,disease and function analysis found that down-regulation of CD59 significantly inhibited cell proliferation.And we have verified through experiments that some of the genes are related to the down-regulation of CD59,so we preliminarily speculate that CyclinD family members and CD59 interact with each other,both of which affect the proliferation of glioma cells.In summary,this study discovered the regulatory effect of complement regulatory protein CD59 on the proliferation of glioma cells,and explored the mechanism of regulation of proliferation,suggesting that CD59 may be used as an immunotherapy target for glioma,which provided a theoretical basis for tumor research and treatment. |
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